Abstract
Integrin dependent regulation of growth factor signalling confers anchorage dependence that is deregulated in cancers. Downstream of integrins and oncogenic Ras the small GTPase Ral is a vital mediator of adhesion dependent trafficking and signalling. This study identifies a novel regulatory crosstalk between Ral and Arf6 that controls Ral function in cells. In re-adherent mouse fibroblasts (MEFs) integrin dependent activation of RalA drives Arf6 activation. Independent of adhesion constitutively active RalA and RalB could both however activate Arf6. This is further conserved in oncogenic H-Ras containing bladder cancer T24 cells, which express anchorage independent active Ral that supports Arf6 activation. Arf6 mediates active Ral-exocyst dependent delivery of raft microdomains to the plasma membrane that supports anchorage independent growth signalling. Accordingly in T24 cells the RalB-Arf6 crosstalk is seen to preferentially regulate anchorage independent Erk signalling. Active Ral we further find uses a Ral-RalBP1-ARNO-Arf6 pathway to mediate Arf6 activation. This study hence identifies Arf6, through this regulatory crosstalk, to be a key downstream mediator of Ral isoform function along adhesion dependent pathways in normal and cancer cells.
Highlights
Anchorage independent signalling in cancer cells is a key component of cancer cell invasion and metastasis, reflecting the ability of cancer cells to survive and grow in diverse and otherwise unfavourable environments
Our previous studies showed that RalA and Arf6 activity decreases on loss of adhesion and recovers upon re-adhesion to fibronectin [19,20]
In defining the regulatory crosstalk between RalA and Arf6 downstream of integrins and oncogenic Ras, this study reveals the role Arf6 has downstream of Ral and how it could help mediate Ral isoform specific function in cells
Summary
Anchorage independent signalling in cancer cells is a key component of cancer cell invasion and metastasis, reflecting the ability of cancer cells to survive and grow in diverse and otherwise unfavourable environments. In Ras-dependent cancers, anchorage independence is induced largely through the RasRalGEF-Ral pathway, in which the Ras related GTPases RalA and RalB are the key mediators [1,2]. Ral GTPases are activated in Ras-independent cancers through other mechanisms [3,4,5].Ral isoforms, RalA and RalB, though 82% identical regulate distinct cellular functions in normal and cancer cells [6,7,8,9]. RalA is implicated in driving anchorage independence [5,10]while RalB supports cell survival in cancers [11]. RalA and RalB bind the same effectors but can signal differently, mediated by their differential activation [6], utilization of downstream effectors [1]and/or localization in cells [8,12,13]
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